The goal of this project is to advance knowledge of mechanisms in synaptic transmission. More specifically, the project will investigate pathways of synaptic vesicle endocytosis and recycling following the collapse of a fused synaptic vesicle into the plasma membrane. While it is well established that clathrin and dynamin- dependent endocytosis plays a major role in this process, key mechanistic aspects of this process remain elusive. In addition, there is evidence for clathrin and dynamin-independent "bulk" endocytic pathways at the synapse, but such pathways remain poorly understood. The role of endosomal compartments in the vesicle cycle also remains controversial. This grant application plans first to characterize endocytic traffic that operates in parallel to the classic clathrin-dependent endocytic pathway and to test the hypothesis that bulk endocytosis leads to the generation of new synaptic vesicles independently of clathrin and of the GTPase dynamin. Second, the project will investigate the properties, potential heterogeneity and interrelationships of endocytic compartments of nerve terminals, determine the function of 3-phosphorylated phosphoinositides in their dynamics and test the hypothesis that most intermediates in synaptic vesicle recycling are not classical endosomes. Third, mechanistic aspects of the clathrin and dynamin-dependent endocytic reaction will be investigated. These studies will involve mouse genetics, studies in cultured neurons and in cell-free systems. This work is of specific relevance in neuroscience, but given the fundamental nature of endocytosis and post- endosomal sorting, it will have broad implications for a variety of fields of biology and medicine. PUBLIC HEALTH RELEVANCE: Synaptic vesicles are the secretory organelles that store and secrete neurotransmitters at neuronal synapses. It is well established that following their fusion with the plasma membrane during neurotransmitter release, synaptic vesicle membranes must be recycled for the generation of new synaptic vesicles, but the precise mechanisms underlying this process remain a matter of debate. The goal of this proposal is to capitalize on recent advances in imaging methods and on perturbations of synaptic vesicle recycling in genetically modified mice and in cells derived from such mice to advance understanding of this fundamental process that supports communication between neurons.